(Magnoliales: Annonaceae) Extracts to Zabrotes Subfasciatus

Lethal and sublethal toxicities of Annona sylvatica (Magnoliales: Annonaceae) extracts to Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) Gabriel Luiz Padoan Gonçalves1, Leandro do Prado Ribeiro2,*, Leila Gimenes3, Paulo Cezar Vieira3, Maria Fátima das Graças Fernandes da Silva3, Moacir Rossi Forim3, João Batista Fernandes3, and José Djair Vendramim1

Abstract Plant secondary metabolites comprise a diverse range of compounds (allelochemicals) that affect insect–plant interactions; many function in plant defense against herbivory. Thus, allelochemicals constitute an important source of insecticidal molecules that potentially can be used in different forms in integrated pest management programs. The objective of this study was to evaluate the bioactivity of ethanolic extracts and partially purified fractions of these extracts obtained from the leaves, branches, and seeds ofAnnona sylvatica A. St.-Hil. (Magnoliales: Annonaceae), a native Brazilian species, against Zabrotes subfasciatus (Boheman) (Coleoptera: Chrysomelidae: Bruchinae). In the screening assay, the ethanolic extract −1 of A. sylvatica seeds was the most promising treatment, causing lethal (LC50 = 753.47 and 701.06 mg kg for males and females, respectively) -−1 and sublethal effects, mainly oviposition deterrence (EC50 = 438.70 mg kg ). On the other hand, ethanolic extracts prepared from branches and −1 leaves caused only sublethal effects including mainly oviposition deterrence (EC50 = 1,168.90 and 1,010.70 mg kg , respectively) and reduction in number of offspring. Based on these results, the extracts were submitted to liquid–liquid partitioning, and their fractions were tested against Z. subfasciatus to verify their bioactivity. Overall, the results of the fraction bioassays showed evidence of synergistic interactions among compounds of different chemical classes and polarities. Chemical analyses of active fractions revealed the presence of triglycerides, alkaloids, and acetogenins in the seed fractions; alkaloids, lignans, and long-chain fatty acid ethyl esters in the branch fractions; and glycosides, flavonoids, terpenoids, and long-chain fatty acid ethyl esters in the leaf fractions. Thus, A. sylvatica is an interesting and potentially important source of structurally diverse grain-protective compounds.

Key Words: Mexican bean weevil; bioactivity; allelochemicals; acetogenins; alkaloids; lignans

Resumo Metabólitos secundários de plantas compreendem uma gama diversificada de compostos (aleloquímicos) que, entre outras funções, atuam na regu- lação das interações inseto-planta e, muitos deles, atuam na defesa da planya contra a herbivoria. Assim, aleloquímicos constituem uma importante fonte de moléculas inseticidas que podem ser utilizadas em programas de manejo integrado de pragas (MIP) em diferentes formas. Portanto, o objetivo deste estudo foi avaliar a bioatividade de extratos etanólicos e frações parcialmente purificadas desses extratos obtidos de folhas, de ramos e de sementes de Annona sylvatica A.St.-Hil. (Magnoliales: Annonaceae), uma espécie nativa do Brasil, sobre Zabrotes subfasciatus (Boheman) (Coleoptera: Chrysomelidae: Bruchinae). Na triagem inicial, o extrato etanólico de sementes foi o tratamento mais promissor, causando efeitos letal −1 (CL50 = 753,47 e 701,06 mg kg , respectivamente, para machos e fêmeas) e subletais, especialmente deterrência de oviposição (EC50 = 438,70 mg kg−1). Por outro lado, os extratos etanólicos preparados a partir de ramos e de folhas causaram apenas efeitos subletais, principalmente deterrência −1 de oviposição (EC50 = 1168,90 e 1010,70 mg kg , respectivamente) e efeito na progênie. Com base nesses resultados, os extratos foram submetidos à partição líquido-líquido e as frações obtidas testadas sobre Z. subfasciatus para verificar sua bioatividade. De um modo geral, os resultados dos bioensaios com as frações mostraram evidências de sinergismo entre compostos de diferentes classes químicas e polaridades. As análises químicas mostraram a presença de triglicerídeos, alcaloides e acetogeninas nas frações ativas do extrato de sementes; alcaloides, lignanas e ésteres etílicos de ácidos graxos de cadeia longa nas frações ativas do extrato de ramos; e glicosídeos, flavonoides, terpenoides e ésteres etílicos de ácidos graxos de cadeia longa nas frações ativas do extrato de folhas. Assim, A. sylvatica é uma fonte interessante de compostos estruturalmente diversificados com propriedades protetoras de grãos.

Palavras Chave: caruncho-do-feijão; bioatividade; aleloquímicos; acetogeninas; alcaloides; lignanas

The development of sustainable practices that can be incorporated Brazil, this is even more evident when taking into consideration that into food production systems currently constitutes one of the great- current pest management programs of major agricultural commodi- est challenges for the scientific community (Prasifka & Gray 2012). In ties are based on the systematic and routine application of synthetic

1Department of Entomology and Acarology, University of São Paulo/ “Luiz de Queiroz” College of Agriculture (USP/ESALQ) – Piracicaba, SP, Brazil 2Research Center for Family Farms, Research and Rural Extension Company of Santa Catarina (CEPAF/EPAGRI) – Chapecó, SC, Brazil 3Department of Chemistry, Federal University of São Carlos (UFSCar) – São Carlos, SP, Brazil *Corresponding author; E-mail: [email protected]; [email protected]

2015 — Florida Entomologist — Volume 98, No. 3 921 922 2015 — Florida Entomologist — Volume 98, No. 3 pesticides instead of an integrated pest management approach, which tus, chemical control has some limitations that are related mainly to takes into account the economic, environmental, and social aspects of the cost of insecticides, which hinders their availability to small-farm pest control interventions (Norris et al. 2003). owners, and the limited number of active ingredients of synthetic in- Recently, with the decreased availability of synthetic molecules secticides available on the Brazilian market. with insecticidal action from the market of compounds with broad- spectrum action, interest in botanical insecticides as an alternative method for insect pest control has been revived (Dayan et al. 2009). Materials and Methods The advantages of botanical insecticides compared with conventional insecticides are related to their lower mammalian toxicity andde- PLANT SAMPLES AND PREPARATION OF CRUDE EXTRACTS creased health risk to applicators and their rapid degradability, which reduces residues in the environment and in the treated products (Is- The plant parts used in the study (branches, leaves, and seeds) man 2006). Additionally, they generally contain various biologically ac- were collected on 23 Mar 2011 from specimens of A. sylvatica grown tive compounds capable of synergistic interactions that may reduce in a domestic orchard located in Erval Seco municipality, RS State, Bra- the selection of resistant pest populations in the field (Akhtar & Isman zil (27°25’41.8”S, 53°34’11.2”W; 466 m asl). A voucher specimen, pre- 2013). Owing to the ease of obtaining botanical derivatives (as plant viously identified by Dr. Renato Mello-Silva (Institute of Biosciences, powders, crude extracts, or oils), these botanical derivatives have been University of Sao Paulo, Brazil), was deposited in the ESA herbarium in used for various purposes in the management of insect pests of stored Piracicaba, SP, Brazil (record number 121205). grains in many countries, especially Latin America, Africa, and Asia (Is- For preparing the extracts, the collected plant structures were man 2008). Another important aspect of research on botanical deriva- dried in an oven at 40 °C for 48 to 72 h. Next, the structures were tives with insecticidal potential is the discovery of model prototypes ground in a knife mill, and the resulting branch (100 g), leaf (100 g), or that can be used in the synthesis of new insecticides, especially those seed (100 g) powders were stored separately in sealed glass contain- with different modes/mechanisms of action and less impact on the en- ers. The organic extracts were prepared by allowing each plant powder vironment and human health (Regnault-Roger et al. 2012). to steep in ethanol (1:5 w/v) in a hermetically sealed flask for 3 d. The Several phytochemicals with insect-associated biological proper- samples were then filtered through filter paper; this procedure was ties are known; these chemicals can act as feeding and oviposition repeated 3 times. The ethanol in the filtered solution was removed in deterrents, repellents, attractants, growth inhibitors, and insecticides a rotary evaporator at 50 °C at a vacuum of −600 mm Hg. After com- (Al Lawati et al. 2002). Brazilian scientists are in a prime position to pleting the evaporation of the solvent in an aerated chamber, the yield identify such active phytochemicals, as plant genetic diversity is higher was determined by weighing the extracts from each A. sylvatica plant in Brazil than in all other countries, with more than 55,000 vascular part, i.e., 5.01, 7.18, and 11.00% (w/w) for the branches, leaves, and plant species catalogued in Brazil (Simões & Schenkel 2002). seeds, respectively. Among botanical families from tropical regions, plants of the family Annonaceae are one of the most promising sources of bioactive BIOASSAYS molecules because of the wide variety of biologically active secondary All bioassays were conducted in a temperature-controlled room at metabolites produced by this family (Leboeuf et al. 1980; Bermejo et 25 ± 2 °C and 60 ± 10% relative humidity with a 14:10 h L:D photope- al. 2005). The acetogenins, a chemically diverse class of compounds riod and a mean illuminance of 200 lux. As the substrate for perform- found in some genera of Annonaceae (Alali et al. 1998), are promising ing the tests, we used shelled bean seeds (P. vulgaris ‘Bolinha’) that compounds that act against insect pests; thus, they may be developed we obtained in the Piracicaba (SP, Brazil) trade and that we manually as new grain protectants (Ribeiro et al. 2013). selected. The tree species Annona sylvatica A. St.-Hil. (Magnoliales: Annona- For the crude extract application on the surface of the bean seeds, ceae), previously assigned to the genus Rollinia, is native to south- a microatomizer coupled to a vacuum pump was used. This microato- central Brazil (Lobão et al. 2005). It produces edible fruits with con- mizer was adjusted to provide a pressure of 0.5 kg cm−2 to deliver a siderable health benefits due to the presence of phenolic compounds, spray volume of 30 L t−1 (30 mL per kg of seeds) according to condi- antioxidants, carotenoids, and vitamin C (Pereira et al. 2013), making tions defined in previous studies (Ribeiro 2010). After spraying, the this species common in domestic orchards. However, despite its rela- bean seeds were transferred to a plastic bag with a capacity of 2 L and tive abundance, not much is known about A. sylvatica from a phyto- were stirred manually for 1 min. Preliminary tests were performed to chemical point of view, especially concerning the bioactive potential of evaluate the homogeneity of the application and to verify the possible its secondary metabolites. To date, sylvaticin, an acetogenin that can effects of the solvents used for extract solubilization onZ. subfasciatus. be isolated from hexane extract of A. sylvatica fruit, is the only allelo- To identify promising bioactive extracts with activity against Z. subfas- chemical reported to have effects on insects. Its action has been shown ciatus, bioassays were conducted to assess the lethal and sublethal on Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) and Acalymma effects (acute and chronic toxicity) of the extracts. vittata F. (Coleoptera: Chrysomelidae) (Mikolajczak et al. 1990). The aim of this study was to evaluate the bioactivity of extracts and semi-purified fractions from different parts (branches, leaves, and EVALUATION OF INSECTICIDAL ACTIVITY seeds) of A. sylvatica on the Mexican bean weevil Zabrotes subfas- In this bioassay, Petri dishes (6 cm diameter × 2 cm height) con- ciatus (Boheman) (Coleoptera: Chrysomelidae: Bruchinae). Zabrotes taining 10 g of beans were used. These sample units were treated subfasciatus is considered to be the main insect pest of stored beans separately with ethanolic extracts of branches, leaves, or seeds at a (Phaseolus vulgaris L.; Fabales: Fabaceae), a staple dietary vegetable in concentration of 1,500 mg kg−1 (1.5 g of extract per kg of bean seeds), many developing countries such as Brazil. In addition to the immeasur- which was determined based on previous studies (Ribeiro et al. 2013). able loss of grain quality, the direct losses caused by this pest species For the control, the bean seeds were treated with only the solvent were estimated to be 35% in Mexico, Central America, and Panama solution (acetone:methanol 1:1 [v/v]) used in the suspension of the and between 7 and 15% in Brazil (Van Schoonhoven & Cardona 1982). extracts. Ten replicates per treatment were used, and each sample unit Despite the relative effectiveness of chemical control of Z. subfascia- was infested with 5 weevil pairs aged between 0 and 24 h that were Gonçalves et al.: Toxicity of Annona sylvatica extracts to Zabrotes subfasciatus 923

derived from a population maintained under laboratory conditions for the respective LC50 estimated for the crude extract, and the same ex- approx. 15 generations. Adult survival was assessed on the 5th day perimental procedures described previously for the screening assay after infestation. Insects with legs completely extended that showed were adopted. For cases where LC50 could not be estimated (> tested no reaction to contact with a thin brush for 1 min of observation were range for extracts of leaves and branches), the fractions were tested considered dead. at the same concentration that was used in the bioassays with the crude extracts (1,500 mg kg−1). In these tests, the same variables and experimental procedures adopted in the previous tests were used, and ASSESSMENT OF SUBLETHAL EFFECTS for each treatment, 10 replicates containing 5 pairs of Z. subfasciatus, The same experimental arrangement used in the previous test was aged between 0 and 24 h, were used. used to evaluate the sublethal effects of the crude extracts. For this assessment, the adults were removed after 5 d of infestation, and the CHEMICAL ANALYSIS OF BIOACTIVE FRACTIONS eggs on each bean seed surface were counted with the aid of a stereo- microscope. Then, the sampling units were maintained under the cli- To identify the class of compounds present in the bioactive fractions, hydrogen nuclear magnetic resonance1 ( H NMR) was performed matic conditions mentioned previously. At 60 d after the initial infesta- 1 tion, the adults that emerged were separated by gender and counted. with a BRUKER DRX 400 instrument operating at 400 MHz for H nucle- us (9.4 Tesla) and the deuterated solvents CDCl and CD OD. At this time, the percentage of damaged seeds (with holes) in each 3 3 sample was determined through visual assessment of each seed. Simi- The chemical profiles of the bioactive fractions were analyzed using lar to the previous bioassay, 10 replicates per treatment were used. thin-layer chromatography (TLC). The TLC analyses were carried out by testing the proportions of solvents of different polarities with the aim of identifying the best mobile phase for the separation of the compo- CONCENTRATION–RESPONSE CURVES OF THE PROMISING nents present in the samples including hexane:dichloromethane 1:1 EXTRACTS (v/v), hexane:acetone 8:2, 7:3, and 1:1 (v/v), dichloromethane:acetone 7:3 and 1:1 (v/v), dichloromethane (100%), and acetone (100%). For The extracts that showed the most promising results were tested the detection of the spots of the constituents present in each sam- for LC and LC estimations, which correspond to the concentrations 50 90 ple, the following tools were used: ultraviolet light (254 and 365 nm), required to kill 50 and 90%, respectively, of the weevil population. vanillin sulfuric solution (general developer), ethanolic solution of 1% Here, preliminary tests were performed using these extracts to deter- aluminum chloride (for detection of flavonoids [Jácome et al. 2010]), mine the basic concentrations that caused 95% adult mortality and a Dragendorff reagent (specific to alkaloids [Sherma 2000]), and Kedde mortality rate similar to the control. Based on these data, a range of reagent (indicating the presence of the g-lactone-α,b-unsaturated sub- concentrations (100 to 2,500 mg kg−1 [mg of extract per kg of seeds]) unit present in acetogenins [Caloprisco et al. 2002]). were set to determine the LC50 values for both males and females, and this was accomplished by applying the formula proposed by Finney (1971). Concentration–response curves were constructed to estimate DATA ANALYSES the EC (the effective concentration required to reduce the number 50 Generalized linear models (GLMs) belonging to the exponential of eggs laid per sample by 50% [oviposition deterrence]). The same family of distributions (Nelder & Wedderbum 1972) were used to as- procedures described previously were used. sess the biological variables of Z. subfasciatus exposed to the extracts and fractions of A. sylvatica. The quality of the fit was verified using LIQUID–LIQUID PARTITIONING OF THE SELECTED CRUDE a half-normal probability graph with a simulation envelope (Hinde & EXTRACTS Demétrio 1998). When significant differences were observed between treatments, multiple comparisons (Tukey test, P < 0.05) were per- Based on the results from the bioassays described previously, the formed using the glht function of the multicomp package with adjusted most promising extracts were selected and subjected to liquid–liquid P values. These analyses were performed using the statistical software partitioning. Specifically, the selected extracts were solubilized sepa- R, version 2.15.1 (R Development Core Team 2012). rately in methanol:water (hydro–methanol fraction; 1:3 [v/v] for the To estimate the lethal concentrations (LC50 and LC90), we used a leaf and branch extracts and 8:2 [v/v] for the seed extracts) and par- binomial model with a complementary log–log link function (gompit titioned in a separatory funnel, using hexane for the seed extract and model), using the Probit Procedure of SAS software, version 9.2 (SAS organic solvents of increasing polarity (hexane, dichloromethane, and Institute 2013). To estimate the average effective concentration (EC50), ethyl acetate) for the leaf and branch extracts. The fractions were con- i.e., the concentration required to reduce the number of eggs per sam- centrated on a rotary evaporator at 50 °C at a vacuum of −600 mm Hg. ple by 50%, we employed a non-linear logistic model using the Nlin The yields were determined using the mass of the respective extracts Procedure of SAS software version 9.2 (SAS Institute 2013). Finally, the as the basis. The seed fractions yielded 83.93 and 16.07% (w/w) for the mean lethal time (LT50) was estimated using the method proposed by hexane and hydro–methanol, respectively; leaf fractions yielded 41.76, Throne et al. (1995) for the Probit analysis of correlated data. 3.88, 5.98, and 48.38% for the hexane, dichloromethane, ethyl acetate, and hydro–methanol, respectively; and branch fractions yielded 16.59, 8.54, 6.87, and 68.00% for the hexane, dichloromethane, ethyl acetate, Results and hydro–methanol, respectively. BIOLOGICAL ACTIVITY OF ETHANOLIC CRUDE EXTRACTS ASSESSMENT OF THE BIOACTIVITY OF THE OBTAINED The ethanolic extract from A. sylvatica seeds was the only treat- FRACTIONS ment that caused significant mortality of Z. subfasciatus adults (Table Each of the obtained fractions (phases) was tested to evaluate its 1). There was no significant difference in mortality between the sexes −1 2 lethal and sublethal effects on Z. subfasciatus. In this assessment, the (LC50 for females: 701.06 mg kg [CI 95%: 664.85–727.67], χ = 2.21, df = −1 2 sample units (10 g of beans) were treated with these fractions using 5; LC50 for males: 753.47 mg kg [CI 95%: 636.53–804.31), χ = 10.36, df 924 2015 — Florida Entomologist — Volume 98, No. 3

Table 1. Mortality (mean ± se) of Zabrotes subfasciatus adults exposed to samples of bean seeds treated with crude ethanolic extracts from different Annona sylvatica parts (at 1,500 mg kg−1) after they had been pulverized and steeped for 5 d in ethanol.

Mortality (%)a

A. sylvatica plant parts Males Females Total Leaves 4.00 ± 2.66 b 8.00 ± 4.42 b 6.00 ± 2.66 b Branches 4.00 ± 4.00 b 4.00 ± 2.66 b 4.00 ± 2.21 b Seeds 100.00 ± 0.00 a 100.00 ± 0.00 a 100.00 ± 0.00 a Control (acetone:methanol 1:1 [v/v]) 2.00 ± 2.00 b 2.00 ± 2.00 b 2.00 ± 2.00 b F value 63.49 75.15 145.61 P value < 0.0001 < 0.0001 < 0.0001

aMeans followed by different letters within columns indicate significant differences between treatments (GLM with a quasi-binomial distribution followed by Tukey’s post hoc test, P < 0.05).

= 5). Moreover, the average lethal time LT50 did not differ between the pecially the hexane fraction, in both cases) caused a significant reduc- 2 sexes (males: 27.59 h [CI 95%: 24.89–30.10], χ = 6.44, df = 8; females: tion in the number of eggs per sample and the number of F1 progeny, 28.81 h [CI 95%: 25.98–31.48], χ2 = 7.80, df = 8). and in the damage caused to the treated bean samples (Table 4). More- The ethanolic extracts prepared from the 3 plant parts caused over, the hydro–methanol and hexane fractions from the partitioning significant sublethal effects (Table 2), mainly a large reduction in the of the ethanolic extract of A. sylvatica seeds significantly affected all −1 number of eggs per sample (EC50: 1,010.70 mg kg [CI 95%: 643.10– parameters (Table 4). However, there was no difference in effects be- 1,378.40], 1,168.90 mg kg−1 [CI 95%: 832.80–1,505.90], and 438.70 tween these 2 fractions, except for the percentage of damaged seeds, mg kg−1 [CI 95%: 403.80–473.60] for the ethanolic extracts from the where the hydro–methanol fraction reduced the damage caused to the leaves, branches, and seeds, respectively). These effects were accord- treated bean samples to a much greater extent than the hexane frac- ingly reflected in the F1 progeny size and the percentage of damaged tion of the ethanolic seed extract. seeds, i.e., variables that can significantly influence the population- dy namics of pest species and the level of degradation of seed quality. However, the extracts had no effect on the egg-to-adult viability or the CHEMICAL ANALYSIS OF BIOACTIVE FRACTIONS sex ratio of the F1 progeny (Table 2), demonstrating that the bioactive The analysis of the hydro–methanol fraction of the seeds using compounds in ethanolic extracts had no effect on the embryonic and TLC (hexane:acetone 7:3 [v/v]) reacted positively with Kedde reagent post-embryonic development of Z. subfasciatus. (spots with a reddish color), as brown spots appeared after the use of sulfuric vanillin solution, indicating the presence of acetogenins. The BIOLOGICAL ACTIVITIES OF THE FRACTIONS OBTAINED FROM analysis using Dragendorff reagent revealed orange spots at the base of the analytical plate, indicating the presence of polar alkaloids in this ETHANOLIC EXTRACTS fraction, and absorption occurred at 365 nm after visualization using None of the fractions originating from the ethanolic extracts pre- UV light (confirming the presence of compounds with a high conjuga- pared from the branches and leaves of A. sylvatica caused significant tion of π bonds, observed in the alkaloids). This fraction was analyzed acute toxicity in adult Z. subfasciatus (Table 3). However, the hexane using 1H NMR, which allowed the identification of the major classes and hydro–methanol fractions of the crude seed extract caused signifi- of compounds present in this fraction as acetogenins. Thus, dH 7,28, cant adult mortality, and there was no difference in mortality (P > 0.05) dH 5,05, and dH 1,38 refer to the lactone α,b-unsaturated unit, signals between the sexes (Table 3). Based on the mortality levels caused by at the region of dH 4,42 to dH 3,08 are related to the oxymethine hy- the partially purified fraction in comparison with the ethanolic extract drogens of their substituents, and signals at dH 2,49 and dH 2,37 refer (Table 3), it was possible to infer that the insecticidal activity of the to diastereotopic hydrogens adjacent to the lactone ring (Cortes et al. crude seed extract was due to the interaction among compounds with 1993; Colman-Saizarbitoria et al. 1995). low and high polarity. The hexane fraction of the ethanolic seed extract, present The hexane and ethyl acetate fractions from the partitioning of the as an oil at room temperature, was similarly analyzed using TLC leaf ethanolic extract and the dichloromethane and hexane fractions (hexane:dichloromethane 1:1 [v/v]) and developed using a vanillin/ from the partitioning of the branch ethanolic extract ofA. sylvatica (es- sulfuric acid solution; the appearance of blue spots indicated the pres-

Table 2. Sublethal effects (mean ± se) of crude ethanolic extracts from different parts of Annona sylvatica at 1,500 mg kg−1 on Zabrotes subfasciatus.

a F1 progeny No. Viability (%) Sex Grains A. sylvatica plant parts eggs/samplea Males Females Total (egg–adult)b ratioc damaged (%)b Leaves 11.90 ± 2.71 c 5.00 ± 1.08 c 5.10 ± 1.28 c 10.10 ± 2.33 c 85.60 ± 7.52 0.47 ± 0.04 a 21.11 ± 3.73 c Branches 26.60 ± 3.66 b 10.00 ± 2.02 b 11.60 ± 1.79 b 21.60 ± 3.46 b 77.61 ± 3.72 0.55 ± 0.04 a 36.43 ± 4.53 b Seeds 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d — — 0.00 ± 0.00 d Control (acetone:methanol 1:1 [v/v]) 83.70 ± 5.02 a 37.90 ± 2.03 a 36.90 ± 2.96 a 74.80 ± 4.63 a 89.30 ± 0.84 0.49 ± 0.01 a 90.60 ± 1.87 a F: 72.18 F: 67.62 F: 54.77 F: 70.25 F: 2.62ns χ2: 53.54 F: 89.48 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.0918 P = 0.0455 P < 0.0001

Table 3. Mortality (mean ± SE) of Zabrotes subfasciatus adults exposed to samples of bean seeds treated with fractions prepared by liquid–liquid partitioning from ethanolic extracts of different Annona sylvatica parts after they had been steeped for 5 d in ethanol.

Mortality (%)a

Fractions Males Females Total A. sylvatica leaves

Hexane 4.00 ± 2.67 6.00 ± 4.27 5.00 ± 3.07 Dichloromethane 6.00 ± 3.05 6.00 ± 4.20 6.00 ± 3.09 Ethylacetate 4.00 ± 2.67 8.00 ± 4.42 6.00 ± 2.21 Hydro–methanol 2.00 ± 2.00 8.00 ± 3.27 4.00 ± 1.63 Control (acetone) 8.00 ± 4.42 8.00 ± 4.42 8.00 ± 3.59 Control (methanol) 10.00 ± 4.47 8.00 ± 3.27 9.00 ± 2.77 F value 0.81ns 0.07ns 0.33ns P value 0.5479 0.9968 0.8911 A. sylvatica branches

Hexane 24.00 ± 5.81 a 30.00 ± 5.37 a 27.00 ± 4.95 Dichloromethane 22.00 ± 10.08 a 14.00 ± 6.70 a 18.00 ± 7.27 Ethylacetate 12.00 ± 3.26 a 12.00 ± 4.42 a 12.00 ± 3.26 Hydro–methanol 2.00 ± 2.00 a 28.00 ± 8.54 a 15.00 ± 4.53 Control (acetone) 8.00 ± 4.42 a 8.00 ± 4.42 a 8.00 ± 3.59 Control (methanol) 10.00 ± 4.47 a 8.00 ± 3.27 a 9.00 ± 2.77 F value 2.62 2.78 2.21ns P value 0.0342 0.0263 0.0668 A. sylvatica seeds

Hexane 30.00 ± 9.45 a 6.00 ± 2.89 ab 15.00 ± 5.96 a Hydro–methanol 44.00 ± 12.22 a 18.00 ± 3.59 a 31.00 ± 6.57 a Control (acetone:methanol 1:1 [v/v]) 2.00 ± 2.00 b 4.00 ± 2.67 b 3.00 ± 1.53 b F value 16.32 4.40 17.11 P value < 0.0001 0.0221 < 0.0001

aMeans followed by different letters within columns (each plant part) indicate significant differences between treatments (GLM with a quasi-binomial distribution followed by Tukey’s post hoc test, P < 0.05); nsNon-significant (P > 0.05). ence of triglycerides. These compounds are the major constituents of Due to the non-polar nature of the hexane fractions from the leaves vegetable seed oils, which are also classified as triacylglycerols, where- and branches, their chromatographic profiles were analyzed using TLC in each functional ester group contains a saturated or unsaturated hy- and 1H NMR. The TLC analysis allowed the comparison of the chemical drocarbon chain (Fernandes et al. 2002). The presence of these com- profiles of both fractions, which were characterized by the presence of pounds in this fraction was confirmed by the analysis of signals in the spots with the same retention factor for both samples (indicating the 1H NMR spectrum, which presented a signal for this class of compound similarity of compounds present in both fractions) after elution with as observed by Colzato et al. (2008). hexane:acetone 8:2 (v/v) and staining with vanillin/sulfuric acid solu- The dichloromethane fraction of the branches was eluted in tion. The analysis of the 1H NMR spectrum showed signals of chemi- dichloromethane:acetone 7:3 (v/v), and after the staining of the chro- cal shifts that were characteristic of furofuranlignans, as described by matographic plate with Dragendorff reagent, orange spots that inter- Biavatti et al. (2001). Additionally, signals similar to those described acted strongly with the silica (retention factor: intermediate to high) for the hexane fraction of seeds were observed, which indicated the could be visualized, indicating the presence of alkaloids as constituents presence of triacylglycerols (Colzato et al. 2008). of this fraction. This evidence was confirmed by the analysis ofthe signals observed in the 1H NMR spectrum, showing the characteristic signs of lignans and alkaloids as observed in the literature (Tantisewie Discussion et al. 1989; Biavatti et al. 2001; Pinheiro et al. 2009). The ethyl acetate fraction of the leaves showed a strong interaction Plants synthesize and accumulate large amounts of allelochemi- with the stationary phase of the chromatographic plate. This fraction cals, and concentrate a wide variety of them in their seeds as an was eluted with acetone:methanol 9:1 (v/v) and showed brown spots evolutionary strategy to ensure the survival of the progeny and the after staining with vanillin/sulfuric acid and a methanolic solution of success of the species (Wink 2003). In an evolutionary context, her- aluminum chloride (1%). Yellow spots were observed after UV (365 bivorous insects have constituted major selective pressure and are the nm) analysis, indicating the presence of flavonoids. The 1H NMR spec- main targets of plant secondary metabolism. In addition to the large trum showed signals with different values of chemical shifts, which accumulation in seeds of allelochemicals with insecticidal properties indicated the presence of terpenoids and glycosyl-flavonoids in this (triglycerides, acetogenins, and alkaloids), A. sylvatica synthesizes and fraction as suggested by Ibrahim et al. (2007), Silva et al. (2012), and accumulates a wide variety of allelochemicals in other plant parts (e.g., Somanawat et al. (2012). long-chain fatty acid ethyl esters, lignans, flavonoids, and terpenoids in 926 2015 — Florida Entomologist — Volume 98, No. 3

Table 4. Sublethal effects (mean ± sE) on Zabrotes subfasciatus of fractions prepared by liquid–liquid partitioning from ethanolic extracts of different parts ofAn- nona sylvatica at 1,500 mg kg−1.

a F1 progeny No. eggs/ Viability (%) Sex Grains Fraction samplea Males Females Total (egg–adult)b ratioc damaged (%)b

A. sylvatica leaves

Hexane 1.20 ± 0.72 c 0.60 ± 0.43 d 0.60 ± 0.34 c 1.20 ± 0.73 c 100.00 ± 0,00d 0.50 ± 0.00d 3.20 ± 1.80 d Dichloromethane 79.60 ± 8.96 a 30.40 ± 3.78 b 34.10 ± 3.77 a 64.50 ± 6.92 a 82.15 ± 2.21 0.53 ± 0.03 66.75 ± 4.64 b Ethyl acetate 27.90 ± 7.32 b 9.40 ± 2.53 c 11.30 ± 3.36 b 20.70 ± 5.77 b 72.08 ± 4.82 0.42 ± 0.08 32.17 ± 7.73 c Hydro–methanol 100.40 ± 6.70 a 39.50 ± 2.10 ab 40.20 ± 2.39 a 79.70 ± 4.12 a 77.27 ± 1.69 0.50 ± 0.01 92.32 ± 1.60 a Control (acetone) 103.50 ± 7.84 a 44.80 ± 3.96 a 42.30 ± 2.84 a 87.10 ± 6.26 a 84.40 ± 1.32 0.49 ± 0.01 93.16 ± 1.93 a Control (methanol) 82.30 ± 4.90 a 36.90 ± 2.10 ab 36.00 ± 2.84 a 72.90 ± 4.59 a 88.35 ± 2.07 0.48 ± 0.01 89.02 ± 2.79 a F: 48.03 F: 48.26 F: 42.78 F: 50.92 F: 2.14ns χ2: 42.32ns F: 62.61 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.0916 P = 0.3424 P < 0.0001 A. sylvatica branches

Hexane 15.80 ± 4.44 d 5.00 ± 1.48 c 7.40 ± 2.31 a 12.40 ± 3.47 d 81.91 ± 3.99 0.61 ± 0.09d 22.13 ± 4.82 d Dichloromethane 37.90 ± 7.03 c 13.80 ± 2.75 b 16.70 ± 2.60 b 30.50 ± 5.17 c 84.26 ± 3.65 0.55 ± 0.03 49.28 ± 6.75 c Ethyl acetate 83.90 ± 5.87 ab 37.90 ± 3.26 a 36.30 ± 3.13 a 74.20 ± 5.81 ab 88.01 ± 1.50 0.49 ± 0.02 86.23 ± 2.76 ab Hydro–methanol 61.80 ± 9.25 bc 30.20 ± 4.00 a 27.00 ± 4.46 ab 57.20 ± 8.31 b 92.72 ± 1.78 0.45 ± 0.02 68.99 ± 5.67 b Control (acetone) 103.50 ± 7.84 a 44.80 ± 3.96 a 42.30 ± 2.84 a 87.10 ± 6.26 a 84.40 ± 1.32 0.49 ± 0.01 93.16 ± 1.93 a Control (methanol) 82.30 ± 4.90 ab 36.90 ± 2.10 a 36.00 ± 2.84 a 72.90 ± 4.59 ab 88.35 ± 2.07 0.48 ± 0.01 89.02 ± 2.79 a F: 21.94 F: 27.49 F: 17.64 F: 24.97 F: 0.58ns χ2: 35.48ns F: 32.59 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.6749 P = 0.3026 P < 0.0001 A. sylvatica seeds

Hexane 21.50 ± 4.11 b 3.50 ± 0.49 b 5.00 ± 0.70 b 8.50 ± 1.00 b 37.89 ± 3.73 b 0.53 ± 0.04d 62.07 ± 9.78 b Hydro–methanol 11.60 ± 4.57 b 1.30 ± 0.64 b 1.60 ± 0.74 b 2.90 ± 1.33 b 15.93 ± 4.67 b 0.36 ± 0.13d 7.55 ± 3.02 c Control (acetone:methanol 1:1 [v/v]) 72.10 ± 4.86 a 23.10 ± 2.18 a 22.20 ± 2.12 a 45.30 ± 3.87 a 62.52 ± 2.84 a 0.49 ± 0.02 95.00 ± 1.63 a F: 29.45 F: 78.41 F: 59.73 F: 79.12 F: 27.05 — F: 49.32 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 — P < 0.0001

aMeans followed by different letters within columns indicate significant differences between treatments (GLM with a quasi-Poisson distribution followed by Tukey’s post hoc test, P < 0.05). bMeans followed by different letters within columns indicate significant differences between treatments (GLM with a quasi-binomial distribution followed by Tukey’s post hoc test,P < 0.05). cSex ratio can be inferred from the female fraction data, which are presented here. dNot included in the analysis due to a small sample unit. the leaves; alkaloids, lignans, and long-chain fatty acid ethyl esters in Many oil products are used as adjuvants in the application of pesti- the branches) that are capable of causing potent effects onZ. subfasci- cides. They may act as a vehicle that helps the active ingredient come atus, an important pest of stored beans. Thus, extracts from this native into contact with the target (kidney bean) and achieve better coverage species of Brazilian Annonaceae are an interesting potential source of of its surface, thereby assisting the principal compound in exerting its structurally diverse grain-protective compounds. The observed differ- insecticidal action. Therefore, it is possible that triglycerides only help ences between the bioactivities of the various fractions of the ethano- the active ingredients to express their insecticidal effect. However, if lic extracts obtained from different plant parts (leaves, branches, and triglycerides promote the mortality of exposed insects, this may be due seeds) are most likely associated with changes in their chemical pro- to asphyxia caused by the blocking of weevil spiracles (Hewlett 1947). files and may not be specifically due to the effect of the concentration By contrast, if the insecticidal effect is due to the presence of minor of the same active compounds. chemical compounds or compounds derived from the breakdown of Knowledge of interactions between chemical compounds assists triglycerides, then in the latter case, fatty acids probably are respon- in the formulation of insecticides that contain synthetic molecules sible for this bioactivity. A study conducted by Ramsewak et al. (2001) that are analogous to natural compounds, and in the formulation of supports this hypothesis, as these authors identified 5 compounds in botanical insecticides that contain several active ingredients. Based the hexane extract of the seeds of Dirca palustris L. (Malvales: Thyme- on such knowledge, antagonistic interactions can be avoided and syn- laeaceae). Three triglycerides (1,3-dilinoleoyl-2-olein; 1,3-dioleoyl- ergistic interactions may be optimized. Therefore, the use of crude 2-linolein; 1,2,3-trilinolein) showed no bioactivity, but the other 2 extracts may be more efficient than the use of isolated compounds. compounds (oleic acid and linoleic acid) isolated from the methanol In relation to the activity observed for the seed extract ofA. sylvatica, fraction of the hexane extract of the seeds promoted larval mortal- triglycerides may hypothetically act as an adjunct to the acetogenins ity of the 4th instar of Aedes aegypti (L.) (Diptera: Culicidae) and re- present in the crude extract. Alternatively, the triglycerides alone duced the growth of Helicoverpa zea Boddie (Lepidoptera: Noctuidae), may cause biological effects. It is noteworthy that due to the pre- Lymantria dispar L. (Lepidoptera: Lymantriidae), Orgyia leucostigma dominant presence of triglycerides, it was not possible to identify (Smith) (Lepidoptera: Lymantriidae), and Malacosoma disstria Hübner other compounds in the hexane fraction; thus, it is uncertain whether (Lepidoptera: Lasiocampidae) larvae, likely due to the phagodeterrent the insecticidal effect of the hexane fraction was due to the action action of these compounds. These results demonstrate that fatty acids of triglycerides or of minor compounds belonging to other chemical may exert bioactivity on the insects, which was also reported by Fatope classes. et al. (2000). Gonçalves et al.: Toxicity of Annona sylvatica extracts to Zabrotes subfasciatus 927 Similarly, Annonaceae species have the same fatty acids in their References Cited seeds. Egydio & Santos (2011) observed little variation in the fatty acids of various species of Annona (A. crassiflora, A. coriacea, A. montana, A. cherimola, A. squamosa ´ ‘Pink Mammoth’ and A. cherimola ´ A. squa- Akhtar Y, Isman MB. 2013. Plant natural products for pest management: the magic of mixtures, pp. 231-247 In Ishaaya I, Palli SR, Horowitz AR [eds.], mosa ‘Gefner’), and they found palmitic, stearic, oleic, and linoleic acids Advanced Technologies for Managing Insect Pests. Elsevier, Dordrecht, The to be the major components in all species. Chemical characterization Netherlands. of seed fatty acids from A. sylvatica showed the presence of palmitic Al Lawati HT, Azam KM, Deadman ML. 2002. Insecticidal and repellent proper- (20.84%), stearic (4.26%), oleic (54.41%), and linoleic acids (20.49%) (An- ties of subtropical plant extracts against pulse beetle, Callosobruchus chi- drade et al. 2012). Annonaceae species, including A. sylvatica, produce nensis. Journal of Agricultural Science 7: 37-45. Alali FQ, Kaakeh W, Bennet GW, McLaughlin JL. 1998. Annonaceous acetogenins certain fatty acids with insecticidal effects, as reported by Peñaflor et al. as natural pesticides: potent toxicity against insecticide susceptible and re- (2006). Moreover, in the current study, the hexane extract of the seeds sistant German cockroaches (Dictyoptera: Blattellidae). Journal of Economic of A. sylvatica drastically decreased the oviposition ofZ. subfasciatus fe- Entomology 91: 641-649. males. Thus, some fatty acids present inAnnona seeds may deter ovipo- Alali FQ, Liu XX, McLaughlin JL. 1999. Annonaceous acetogenins: recent progress. Journal of Natural Products 62: 504-540. sition. Indeed, Peñaflor et al. (2006) demonstrated that fatty acids that Andrade JMM, Marin R, Apel MA, Raseira MCB, Henriques AT. 2012 Comparison have between 5 and 9 carbon atoms in their chains are capable of affect- of the fatty acid profiles of edible native fruit seeds from southern Brazil. ing the behavior of insects; more specifically, these fatty acids can repel International Journal of Food Properties 15: 815-822. workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae). Bermejo A, Figadère B, Zafra-Polo MC, Barrachina I, Estornell E, Cortes D. 2005. During the evaluation of the experiments investigating the effect of Acetogenins from Annonaceae: recent progress in isolation, synthesis and mechanisms of action. 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(In Portuguese) The authors thank the São Paulo Research Foundation (FAPESP, Leboeuf M, Cavé A, Bhaumik PK, Mukherjee B, Mukherjee R. 1980. The phyto- grants 2010/52638-0 and 2011/23030-7) for their financial support. chemistry of the Annonaceae. Phytochemistry 21: 2783-2813. 928 2015 — Florida Entomologist — Volume 98, No. 3

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